Cortical HCN channels: Function, Trafficking and Plasticity Mala M. Shah Department of Pharmacology, UCL School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX. e-mail: [email protected]
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Abstract The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium ion channels. They are, however, activated by hyperpolarizing potentials and are permeable to cations. Four HCN subunits have been cloned, of which HCN1 and HCN2 subunits are predominantly expressed in the cortex. These subunits are principally located in pyramidal cell dendrites, although they are also found at lower concentrations in the somata of pyramidal neurons as well as other neuron subtypes. HCN channels are actively trafficked to dendrites by binding to the chaperone protein, TRIP8b. Somato-dendritic HCN channels in pyramidal neurons modulate spike firing and synaptic potential integration by influencing the membrane resistance and resting membrane potential. Intriguingly, HCN channels are present in certain cortical axons and synaptic terminals too. Here, they regulate synaptic transmission but the underlying mechanisms appear to vary considerably amongst different synaptic terminals. In conclusion, HCN channels are expressed in multiple neuronal subcellular compartments in the cortex, where they have a diverse and complex effect on neuronal excitability.
Voltage-gated ion channels play a fundamental role in regulating neuronal activity and synaptic transmission. The abundance and biophysical properties of voltage-gated ion This article is protected by copyright. All rights reserved.
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channels varies within neuronal subcellular compartments: axons, dendrites and somata (Lai and Jan 2006; Johnston and Narayanan 2008; Nusser 2009). This variation in localization has a significant impact on neuronal and neural network excitability and thus physiological processes such as learning and memory and patho-physiological conditions such as epilepsy. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that are permeable to Na+ and K+ ions and open at potentials more negative to 50 mV (Pape 1996; Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; Shah, Hammond et al. 2010). These channels, thus, are active at the normal resting membrane potentials (RMP) of most neurons and contribute to depolarizing the RMP. In addition, HCN channels regulate the membrane resistance. By affecting both the RMP and membrane resistance, HCN channels critically influence intrinsic neuronal excitability, synaptic potential integration and neurotransmitter release (Biel, Wahl-Schott et al. 2009; Shah, Hammond et al. 2010). Four HCN subunits (HCN1-4) have so far been cloned (Ludwig, Zong et al. 1998; Santoro, Liu et al. 1998). All four are expressed in the central nervous system (CNS) but their patterns of expression vary. Of these, only HCN1 subunits are abundantly found in the cortex, hippocampus, cerebellum and brain stem (Moosmang, Biel et al. 1999; Notomi and Shigemoto 2004). In contrast, HCN2 subunits are distributed ubiquitously through the CNS, with highest expression levels in the thalamus and brain stem nuclei. HCN3 subunits are expressed at a level low in the CNS and HCN4 subunits are found in selective brain regions such as mitral cell layer of the olfactory bulb (Moosmang, Biel et al. 1999; Notomi and Shigemoto 2004). These subunits can form homomeric or heteromeric channels when expressed in heterologous systems (Chen, Wang et al. 2001; Biel, Wahl-Schott et al. 2009). The activation time constants and steady state voltagedependence of the individual HCN currents differs considerably (Biel, Wahl-Schott et al. 2009). Thus, HCN1 subunits have very fast activation kinetics whilst HCN4 subunits have the slowest kinetics. In addition, all HCN subunits are modulated by cyclic nucleotides, though the extent to which HCN1-4
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currents are modified by these varies considerably. A number of other intracellular signalling molecules such as phosphoinositides and kinases as well as auxiliary subunits such as TPR-containing Rab8b interacting protein (TRIP8b) also affect the biophysical properties and expression of HCN subunits (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; Wahl-Schott and Biel 2009; Shah, Hammond et al. 2010). This diversity in their expression, biophysical properties and modulation by intracellular molecules is, therefore, likely to differentially and dynamically regulate neuronal excitability (for in-depth reviews on HCN channel structure and biophysical properties, please see (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; Wahl-Schott and Biel 2009). In this review, I will focus on the role of HCN channels in determining cortical neuronal excitability. I will also discuss how HCN subunits are trafficked to selective subcellular compartments (axons and dendrites) and how their activity and plasticity affects pyramidal cell dendritic excitability and presynaptic function. .
Somato-dendritic HCN channel function
Electrophysiological studies have revealed that the HCN current, Ih, is greatest in the distal dendrites of cortical and hippocampal pyramidal neurons (Magee 1998; Stuart and Spruston 1998; Williams and Stuart 2000; Berger, Larkum et al. 2001; Shah, Anderson et al. 2004; Huang, Walker et al. 2009) (Fig 1). These findings have been corroborated with immunohistochemical studies showing the abundance of HCN1 and HCN2 subunits in pyramidal cell distal dendrites (Lorincz, Notomi et al. 2002; Notomi and Shigemoto 2004). HCN channels, though, are also located in the somata of some pyramidal neurons (albeit at a lower density than that in distal dendrites), interneuron subtypes and stellate cells (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009). Here, treatment with HCN channel inhibitors augments the input resistance (RN). Since the RMP is also lowered substantially, there is either a reduction or little change in the number of spikes generated in response to depolarizing stimuli (Maccaferri, Mangoni et al. 1993; Maccaferri and McBain 1996;
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Gasparini and DiFrancesco 1997; Magee 1998; Robinson and Siegelbaum 2003; Fransen, Alonso et al. 2004; Shah, Anderson et al. 2004; Aponte, Lien et al. 2006; van Welie, Remme et al. 2006; Nolan, Dudman et al. 2007; Thuault, Malleret et al. 2013). In some neurons, such as the entorhinal cortical stellate cells, HCN channels are activated during the afterhyerpolarization following spikes and thereby affect spike firing patterns (Nolan, Dudman et al. 2007). In contrast, in distal dendrites, where the expression of HCN channels is the greatest, pharmacological blockade of Ih or genetic ablation of HCN results in augmented dendritic excitability despite a hyperpolarized RMP (Magee 1998; Magee 1999; Berger, Larkum et al. 2001; Poolos, Migliore et al. 2002; Shah, Anderson et al. 2004; Huang, Walker et al. 2009)(Fig 1). This is because the increase in membrane resistance following the inhibition of HCN channels in larger in distal dendrites than at the soma such that the change in voltage induced by depolarizing stimuli in the absence of Ih results in spikes despite the RMP being hyperpolarization (Magee 1998; Poolos, Migliore et al. 2002; Shah, Anderson et al. 2004; Huang, Walker et al. 2009)(Fig 1). Changes in membrane resistance will also affect synaptic potential shapes and integration. Indeed, when HCN channel activity is reduced, somato-dendritic excitatory post-synaptic potential (EPSP) amplitudes are greater and their decay slower, leading to enhanced summation of a train of synaptic potantials (Fig 1) (Magee 1998; Magee 1999; Magee 2000; Williams and Stuart 2000; Berger, Larkum et al. 2001; Poolos, Migliore et al. 2002; Nolan, Malleret et al. 2004; Shah, Anderson et al. 2004; Huang, Walker et al. 2009; Sheets, Suter et al. 2011). Thus, trains of EPSPs are more likely to generate action potentials in axons, boosting neuronal excitability (Shah, Anderson et al. 2004). The increased EPSP summation caused by somato-dendritic HCN channel inhibition is, at least in part, due to the enhanced membrane resistance. In distal dendrites, relief of inactivation of T- and N- type Ca2+ channels by RMP hyperpolarization also contributes to enhanced EPSP summation following
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pharmacological block of HCN channels (Tsay, Dudman et al. 2007). Certainly, Ca2+ entry via these Ca2+ channels during dendritic Ca2+ spikes is greater in the presence of HCN channel inhibitors (Tsay, Dudman et al. 2007). It should be noted, though, that certain subthreshold potassium channels such as KV7/M- channels are likely to have a larger impact on EPSP summation in the absence of Ih, if expressed in the same subcellular domain within a particular neuron subtype (George, Abbott et al. 2009). Hence, the overall effect of HCN channels on EPSP summation is likely to depend to a certain extent on which other ion channels are present locally. Somato-dendritic HCN channels affect the integration of inhibitory synaptic inputs (IPSPs) too (Williams and Stuart 2003; Atherton, Kitano et al. 2010; Pavlov, Scimemi et al. 2011). HCN channels are activated by hyperpolarization and their activation during trains of IPSPs serves to limit synaptic hyperpolarization (Williams and Stuart 2003; Atherton, Kitano et al. 2010; Pavlov, Scimemi et al. 2011). In cortical pyramidal cell neurons, distal dendritic Ih enhances dendro-somatic IPSP attenuation and constrains axo-somatic depolarization (Williams and Stuart 2003). Moreover, in certain neurons, the activation of HCN channels during synaptic inhibition restricts de-inactivation of T-type Ca2+ channels and rebound action potential firing (Atherton, Kitano et al. 2010). Therefore, enhanced HCN channel activity during trains of synaptic inhibitory potentials can profoundly alter the state of neurons and their response to subsequent stimuli. Somato-dendritic HCN channels have also been implicated in regulating neuronal oscillations and subthreshold resonance properties of neurons (Fransen, Alonso et al. 2004; van Welie, Remme et al. 2006; Haas, Dorval et al. 2007; Narayanan and Johnston 2007; Nolan, Dudman et al. 2007; Narayanan and Johnston 2008; Hu, Vervaeke et al. 2009; Marcelin, Chauviere et al. 2009; Vaidya and Johnston 2013). In distal dendrites, HCN channels are thought to act as inverse leaky voltage-dependent inductors and thereby act as a
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bandpass filter to allow synaptic inputs of certain frequency to be preferentially transferred to the soma (Narayanan and Johnston 2008; Vaidya and Johnston 2013). Indeed, recent evidence suggests that HCN channels influence signal processing in distal hippocampal CA1 pyramidal dendrites such that slower, theta frequency synaptic inputs get preferentially transferred to the soma even when dendrites receive synaptic inputs at gamma frequency (Vaidya and Johnston 2013). In addition, the inductance property of HCN channels provides an efficient means of synchronization of inputs so that voltage waveforms at the soma are not very sensitive to the location of the synaptic inputs in the dendritic tree (Vaidya and Johnston 2013). Thus, HCN channels modulate somato-dendritic information processing in a variety of ways all of which impact neuronal synaptic integration and activity.
Role of pre-synaptic HCN channels in synaptic release In addition to their location in dendrites, immunohistochemical evidence has suggested that HCN1 subunits are expressed in cortical and hippocampal axons and synaptic terminals of inhibitory and excitatory neurons (Notomi and Shigemoto 2004; Lujan, Albasanz et al. 2005; Bender, Kirschstein et al. 2007; Boyes, Bolam et al. 2007; Huang, Lujan et al. 2011; Huang, Lujan et al. 2012) (Fig 2). In the rodent hippocampus, HCN channels are present in basket cell axons and terminals, where they inhibit synaptic release by as yet an undefined mechanism (Aponte, Lien et al. 2006). HCN1 subunits are also present in immature perforant path axons, which synapse onto dentate gyrus granule cells (Bender, Kirschstein et al. 2007). Here, interestingly, pharmacological inhibitors of HCN channels moderately reduced short term depression of long trains of EPSPs at 20 Hz but not at lower frequencies. These findings suggested that HCN1 subunits in immature perforant path axons form functional channels, which modulate action potential dependent release (Bender, Kirschstein et al. 2007).
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In adult neurons, the influence of HCN channels on synaptic transmission has so far only been found in the entorhinal cortex (EC). Here, immunogold labeling showed that HCN1 subunits were preferentially localized to the active zone in assymetric (presumably glutamatergic) synaptic terminals (Huang, Lujan et al. 2011; Huang, Lujan et al. 2012) (Fig 2). These terminals also contained the T-type Ca2+ channel subunits, CaV3.2, as evident from co-labelling for HCN1 and CaV3.2 subunits (Huang, Lujan et al. 2011). Pharmacological inhibition or genetic ablation of HCN1 channels resulted in enhanced frequency of miniature excitatory post-synaptic currents (mEPSCs) selectively onto EC layer III pyramidal neurons, suggesting that pre-synaptic HCN channels regulate basal glutamatergic synaptic release (Huang, Lujan et al. 2011) (Fig 2). The significantly increased mEPSC frequency was a consequence of relief of inactivation of CaV3.2 channels caused by membrane hyperpolarization when HCN channel function was reduced (Huang, Lujan et al. 2011). Interestingly, the paired pulse ratio of EPSCs evoked by stimulating distal dendrites of EC layer III pyramidal neurons was significantly lowered in the presence of HCN channel blockers, suggesting that action potential driven release onto EC layer III pyramidal neurons is also regulated by HCN channels (Huang, Lujan et al. 2011). Hence, HCN channels affect multiple modes of synaptic transmission onto EC layer III pyramidal neurons.
Dendritic and pre-synaptic HCN1 subunit trafficking. HCN channels are actively trafficked to dendrites by binding to chaperone proteins known as TPR-containing Rab8b interacting protein (TRIP8b) (Santoro, Wainger et al. 2004; Lewis, Schwartz et al. 2009; Santoro, Piskorowski et al. 2009; Zolles, Wenzel et al. 2009; Shah, Hammond et al. 2010). Moreover, TRIP8b is essential for membrane expression of HCN channels in hippocampal and cortical dendrites (Santoro, Wainger et al. 2004; Lewis, Schwartz et al. 2009; Santoro, Piskorowski et al. 2009). In addition, the presence of TRIP8b
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shifts the activation curve of Ih to the left (Lewis, Schwartz et al. 2009; Santoro, Piskorowski et al. 2009; Zolles, Wenzel et al. 2009). Moreover, the presence of TRIP8b alters sensitivity of HCN channels to cyclic nucleotides (Zolles, Wenzel et al. 2009; Han, Noam et al. 2011; Hu, Santoro et al. 2013). Thus, the presence of TRIP8b is important for the expression and voltage-dependence of dendritic HCN channels. Nine isoforms of TRIP8b have so far been identified, most of which enhance expression of dendritic HCN subunits (Lewis, Schwartz et al. 2009; Santoro, Piskorowski et al. 2009). Some TRIP8b isoforms, however, suppress HCN subunit expression (Zolles, Wenzel et al. 2009; Lewis and Chetkovich 2011; Santoro, Hu et al. 2011). Interestingly, TRIP8b isoforms that hinder HCN subunit expression have been suggested to predominantly exist in adult principal cell (including pyramidal neuron) axons (Piskorowski, Santoro et al. 2011). It has, thus, been proposed that TRIP8b prevents mislocalization of HCN subunits to adult axons (Piskorowski, Santoro et al. 2011). In agreement with this notion, HCN1 subunits are localized to immature perforant path axons when TRIP8b expression is low, suggesting that upregulation of TRIP8b expression during development leads to reduced axonal HCN subunit expression (Wilkars, Liu et al. 2012). Moreover, absence of all TRIP8b isoforms results in HCN1 expression in adult perforant path axons (Wilkars, Liu et al. 2012). HCN1 subunits, however, are expressed in certain adult axons and synaptic terminals in the entorhinal cortex (EC) (Huang, Lujan et al. 2011; Huang, Lujan et al. 2012). Are TRIP8b isoforms, therefore, not localized to these? There was no difference in HCN1 subunit expression in these axons and synaptic terminals when wildtype and TRIP8b tissue was compared (Huang, Lujan et al. 2012). Further, functional pre-synaptic HCN1 channels regulated synaptic release onto EC layer III pyramidal neurons to a similar extent in both wildtype and TRIP8b null mice, suggesting that TRIP8b is not involved in the targeting of HCN1 subunits to these axons (Huang, Lujan et al. 2012). In heterologous systems as well as
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neurons, other proteins such as filamin A have been shown to regulate HCN subunit expression (Biel, Wahl-Schott et al. 2009). It remains to be determined if these proteins might be involved in the targeting and stabilization of HCN subunits in adult axons.
Dendritic and pre-synaptic HCN channel plasticity under physiological and pathophysiological states HCN channel activity in hippocampal CA1 pyramidal neurons is modified by activitydependent mechanisms involving changes in intracellular Ca2+ concentrations (van Welie, van Hooft et al. 2004). Hebbian plasticity including NMDA-receptor dependent long-term potentiation (LTP) also alters dendritic HCN subunit expression and channel function in hippocampal CA1 pyramidal neurons (Shah, Hammond et al. 2010). In CA1 pyramidal neurons, induction of NMDA-receptor dependent LTP via a theta burst protocol enhances HCN channel expression by activating calcium/calmodulin dependent protein kinase II (CaMKII) (Fan, Fricker et al. 2005; Campanac, Daoudal et al. 2008). LTP induced by high frequency stimulation, though, reduces dendritic HCN channel function and causes greater synaptic potential summation and EPSP-spike coupling (Campanac, Daoudal et al. 2008). Further, activation of the 2 adrenoreceptors in prefrontal cortical dendritic spines led to enhanced LTP and working memory via a decrease in spine cAMP and HCN1 channel activity (Wang, Ramos et al. 2007). Alterations in modifications in Ih induced by stimulation of schaffer collateral inputs to CA1 pyramidal neurons were absent in TRIP8b null neurons, indicating changes in TRIP8b expression or function might underlie plasticity induced changes in Ih (Brager, Lewis et al. 2013). Interestingly, though, pairing of schaffer collateral and perforant path inputs produced LTP in TRIP8b null CA1 pyramidal neurons (Brager, Lewis et al. 2013). LTP induced by stimulation of the distal perforant path was also greater in HCN1 null CA1 pyramidal neurons (Nolan, Malleret et al. 2004). Consistent with this, HCN1
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null mice have increased hippocampal dependent learning and memory (Nolan, Malleret et al. 2004). In addition to LTP, induction of long-term depression (LTD) modulates Ih. Thus, metabotropic glutamate receptor-dependent LTD lowered dendritic HCN channel activity due to Ca2+ release from internal stores and activation of PKC (Brager and Johnston 2007). Therefore, whilst activity and Hebbian forms of plasticity modulate dendritic HCN channel function and expression, the consequent change in HCN channel activity (up- or downregulation) is likely to be dependent upon the locus where plasticity is induced and the intracellular signalling cascades triggered by plasticity-inducing mechanisms. Abnormal neuronal activity is a common feature of many neurological disorders too. Indeed, modifications in HCN channel expression and function have been associated with disorders such as neuropathic pain and epilepsy (Biel, Wahl-Schott et al. 2009; Noam, Bernard et al. 2011). Considerable evidence for alterations in HCN channel function has been reported in animal models of epilepsy, particularly those mimicking temporal lobe epilepsy (TLE). TLE is the most common, drug-resistant form of acquired epilepsy (Engel 1996). It can be mimicked in animals by administering chemoconvulsants such as kainic acid or pilocarpine (White 2002). Intriguingly, cortical dendritic HCN1 and HCN2 subunit expression and activity is reduced within hours of chemoconvulsant-induced status epilepticus and remains persistently downregulated for weeks (Shah, Anderson et al. 2004; Jung, Jones et al. 2007; Shin, Brager et al. 2008; Marcelin, Chauviere et al. 2009; Jung, Warner et al. 2011). There is also a long-term decrease in cortical pre-synaptic HCN channel activity following kainic acid induced status epilepticus (Huang, Lujan et al. 2012). Further, a decrease in HCN1 mRNA and current function has also been found in cortical and hippocampal tissue obtained from TLE patients (Bender, Soleymani et al. 2003; Wierschke, Lehmann et al. 2010). HCN2 mRNA levels, though, appear slightly enhanced in epileptic human hippocampus (Bender, Soleymani et al. 2003). Interestingly, though, HCN2 null mice
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have absence epilepsy (Ludwig, Budde et al. 2003) and loss of function mutations in HCN2 subunits have been associated with idiopathic generalized epilepsies in patients (DiFrancesco, Barbuti et al. 2011). HCN1 null mice, however, are not spontaneously epileptic but are more susceptible to chemoconvulsant-induced status epilepticus or kindling. (Huang, Walker et al. 2009; Santoro, Lee et al. 2010) These results suggest that the loss of HCN subunits following status epilepticus is likely to contribute to the process of epileptogenesis. The sustained reduction in HCN subunits has been attributed to multiple mechanisms including repressed transcription of HCN1 by upregulation of Neuron-Restrictive Silencer Factor (NRSF) (McClelland, Flynn et al. 2011) and altered activity of the phosphatase, calcineurin, and the kinase, p38 MAPK, resulting in a leftward shift in the HCN current activation curve and fewer HCN channels available at rest (Jung, Bullis et al. 2010). Indeed, transiently restoring HCN channel expression by disrupting the interaction between the NRSF and HCN1 delays the onset of spontaneous seizure activity following termination of SE (McClelland, Flynn et al. 2011). Intriguingly, HCN2 channel expression and function is increased in hippocampal pyramidal neurons following febrile seizures (Chen, Aradi et al. 2001; Brewster, Bender et al. 2002; Dyhrfjeld-Johnsen, Morgan et al. 2008). A heterozygous missense mutation in HCN2 (S126L) has also been correlated with increased incidence of febrile seizures (Nakamura, Shi et al. 2013). Expressed HCN2 channels containing this mutation displayed faster kinetics with higher temperatures, resulting in enhanced availability of the current under these conditions (Nakamura, Shi et al. 2013). Moreover, dendritic HCN current is enhanced in hippocampal pyramidal neurons in mice in with targeted deletions in the Fragile X FMR1 gene(Brager, Akhavan et al. 2012). FMR1 knockout mice do not exhibit spontaneous seizures but are more susceptible to audiogenic seizures (Musumeci, Bosco et al. 2000). Similarly, only about a third of the rodents subjected to febrile seizures develop chronic epilepsy
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(Walker and Kullmann 1999; Dube, Brewster et al. 2007). Hence, whether HCN channel upregulation under these conditions is a homeostatic change or cause of the epileptogenic activity remains to be further investigated.
Concluding remarks In summary, in the cortex, HCN channels are predominantly located in pyramidal neuron distal dendrites (Johnston and Narayanan 2008; Nusser 2009; Shah, Hammond et al. 2010). They are actively trafficked here by binding to TRIP8b (Shah, Hammond et al. 2010). Dendritic HCN channels lower the membrane resistance and depolarize the resting membrane potential, limiting calcium channel activation. Consequently, inhibition of these channels enhances dendritic excitability and synaptic potential summation despite the membrane potential being hyperpolarized (Robinson and Siegelbaum 2003; Shah, Hammond et al. 2010). Further, dendritic HCN channels contribute to the synchronization of synaptic potentials such that the voltage waveform at the soma is not influenced by the location of the synaptic inputs (Vaidya and Johnston 2013). In addition to TRIP8b, dendritic HCN channels are modulated by a number of different intracellular molecules including cyclic nucleotides, kinases and phosphatases (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; Shah, Hammond et al. 2010). Thus, Hebbian and homeostatic plasticity inducing mechanisms as well as pathological conditions that results in alterations of the activity of various intracellular substances modulate HCN channel function and neuronal excitability. Whilst HCN channel function in cortical pyramidal cell dendrites have been studied the most, HCN channels are also located at the somata of some pyramidal neurons, interneurons and stellate cells (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; WahlSchott and Biel 2009) as well as in a subset of excitatory and inhibitory synaptic terminals
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(Notomi and Shigemoto 2004; Lujan, Albasanz et al. 2005; Bender, Kirschstein et al. 2007; Huang, Lujan et al. 2011). At the somata, HCN channels contribute to maintaining intrinsic excitability too. However, in contrast to their effects in dendrites, pharmacological block or genetic deletion of HCN channels at pyramidal neuron, interneuron or stellate cell somata has little effect on spike firing (Maccaferri, Mangoni et al. 1993; Maccaferri and McBain 1996; Gasparini and DiFrancesco 1997; Magee 1998; Robinson and Siegelbaum 2003; Fransen, Alonso et al. 2004; Shah, Anderson et al. 2004; Aponte, Lien et al. 2006; van Welie, Remme et al. 2006; Nolan, Dudman et al. 2007; Thuault, Malleret et al. 2013). Synaptic potential integration at the soma is affected by HCN channels as well but the overall effect on somatic excitability is likely to depend on the complement of other ion channels expressed. Similarly, HCN channels in synaptic terminals regulate synaptic release but the overall effect in the change in neurotransmission appears to be dependent on which other ion channels might also be located in particular synaptic terminals (Aponte, Lien et al. 2006; Bender, Kirschstein et al. 2007; Huang, Lujan et al. 2011; Huang, Lujan et al. 2012). In comparison to dendritic HCN channels, though, relatively little is known about the regulation, modulation and trafficking of pre-synaptic HCN channels. In summary, HCN channels are diversely expressed within the cerebral cortex, where they contribute to maintaining intrinsic neuronal activity and synaptic transmission, thereby serving to dynamically influencing cortical network activity.
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Ludwig, A., X. Zong, et al. (1998). A family of hyperpolarization-activated mammalian cation channels. Nature 393(6685): 587-591. Lujan, R., J. L. Albasanz, et al. (2005). Preferential localization of the hyperpolarizationactivated cyclic nucleotide-gated cation channel subunit HCN1 in basket cell terminals of the rat cerebellum. Eur J Neurosci 21(8): 2073-2082. Maccaferri, G., M. Mangoni, et al. (1993). Properties of the hyperpolarization-activated current in rat hippocampal CA1 pyramidal cells. J Neurophysiol 69(6): 2129-2136. Maccaferri, G. and C. J. McBain (1996). The hyperpolarization-activated current (Ih) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones. J Physiol 497 ( Pt 1): 119-130. Magee, J. C. (1998). Dendritic hyperpolarization-activated currents modify the integrative properties of hippocampal CA1 pyramidal neurons. J Neurosci 18(19): 7613-7624. Magee, J. C. (1999). Dendritic lh normalizes temporal summation in hippocampal CA1 neurons. Nat Neurosci 2(6): 508-514. Magee, J. C. (2000). Dendritic integration of excitatory synaptic input. Nat Rev Neurosci 1(3): 181-190. Marcelin, B., L. Chauviere, et al. (2009). "h channel-dependent deficit of theta oscillation resonance and phase shift in temporal lobe epilepsy." Neurobiol Dis 33(3): 436-447. McClelland, S., C. Flynn, et al. (2011). Neuron-restrictive silencer factor-mediated hyperpolarization-activated cyclic nucleotide gated channelopathy in experimental temporal lobe epilepsy. Ann Neurol 70(3): 454-464. Moosmang, S., M. Biel, et al. (1999). Differential distribution of four hyperpolarizationactivated cation channels in mouse brain. Biol Chem 380(7-8): 975-980. Musumeci, S. A., P. Bosco, et al. (2000). Audiogenic seizures susceptibility in transgenic mice with fragile X syndrome. Epilepsia 41(1): 19-23. Nakamura, Y., X. Shi, et al. (2013). Novel HCN2 mutation contributes to febrile seizures by shifting the channel's kinetics in a temperature-dependent manner. PLoS One 8(12): e80376. Narayanan, R. and D. Johnston (2007). Long-term potentiation in rat hippocampal neurons is accompanied by spatially widespread changes in intrinsic oscillatory dynamics and excitability. Neuron 56(6): 1061-1075. Narayanan, R. and D. Johnston (2008). The h channel mediates location dependence and plasticity of intrinsic phase response in rat hippocampal neurons. J Neurosci 28(22): 5846-5860. Noam, Y., C. Bernard, et al. (2011). Towards an integrated view of HCN channel role in epilepsy. Curr Opin Neurobiol 21(6): 873-879. Nolan, M. F., J. T. Dudman, et al. (2007). HCN1 channels control resting and active integrative properties of stellate cells from layer II of the entorhinal cortex. J Neurosci 27(46): 12440-12451. Nolan, M. F., G. Malleret, et al. (2004). A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons. Cell 119(5): 719-732. Notomi, T. and R. Shigemoto (2004). Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain. J Comp Neurol 471(3): 241-276. Nusser, Z. (2009). Variability in the subcellular distribution of ion channels increases neuronal diversity. Trends Neurosci 32(5): 267-274. Pape, H. C. (1996). Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu Rev Physiol 58: 299-327. Pavlov, I., A. Scimemi, et al. (2011). I(h)-mediated depolarization enhances the temporal precision of neuronal integration. Nat Commun 2: 199. This article is protected by copyright. All rights reserved.
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Piskorowski, R., B. Santoro, et al. (2011). TRIP8b splice forms act in concert to regulate the localization and expression of HCN1 channels in CA1 pyramidal neurons. Neuron 70(3): 495-509. Poolos, N. P., M. Migliore, et al. (2002). Pharmacological upregulation of h-channels reduces the excitability of pyramidal neuron dendrites. Nat Neurosci 5(8): 767-774. Robinson, R. B. and S. A. Siegelbaum (2003). Hyperpolarization-activated cation currents: from molecules to physiological function. Annu Rev Physiol 65: 453-480. Santoro, B., L. Hu, et al. (2011). TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites. J Neurosci 31(11): 4074-4086. Santoro, B., J. Y. Lee, et al. (2010). Increased seizure severity and seizure-related death in mice lacking HCN1 channels. Epilepsia 51(8): 1624-1627. Santoro, B., D. T. Liu, et al. (1998). Identification of a gene encoding a hyperpolarizationactivated pacemaker channel of brain. Cell 93(5): 717-729. Santoro, B., R. A. Piskorowski, et al. (2009). TRIP8b splice variants form a family of auxiliary subunits that regulate gating and trafficking of HCN channels in the brain. Neuron 62(6): 802-813. Santoro, B., B. J. Wainger, et al. (2004). Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction. J Neurosci 24(47): 10750-10762. Shah, M. M., A. E. Anderson, et al. (2004). Seizure-induced plasticity of h channels in entorhinal cortical layer III pyramidal neurons. Neuron 44(3): 495-508. Shah, M. M., R. S. Hammond, et al. (2010). Dendritic ion channel trafficking and plasticity. Trends Neurosci 33(7): 307-316. Sheets, P. L., B. A. Suter, et al. (2011). Corticospinal-specific HCN expression in mouse motor cortex: I(h)-dependent synaptic integration as a candidate microcircuit mechanism involved in motor control. J Neurophysiol 106(5): 2216-2231. Shin, M., D. Brager, et al. (2008). Mislocalization of h channel subunits underlies h channelopathy in temporal lobe epilepsy. Neurobiol Dis 32(1): 26-36. Stuart, G. and N. Spruston (1998). Determinants of voltage attenuation in neocortical pyramidal neuron dendrites. J Neurosci 18(10): 3501-3510. Thuault, S. J., G. Malleret, et al. (2013). Prefrontal cortex HCN1 channels enable intrinsic persistent neural firing and executive memory function. J Neurosci 33(34): 1358313599. Tsay, D., J. T. Dudman, et al. (2007). HCN1 channels constrain synaptically evoked Ca2+ spikes in distal dendrites of CA1 pyramidal neurons. Neuron 56(6): 1076-1089. Vaidya, S. P. and D. Johnston (2013). Temporal synchrony and gamma-to-theta power conversion in the dendrites of CA1 pyramidal neurons. Nat Neurosci 16(12): 18121820. van Welie, I., M. W. Remme, et al. (2006). Different levels of Ih determine distinct temporal integration in bursting and regular-spiking neurons in rat subiculum. J Physiol 576(Pt 1): 203-214. van Welie, I., J. A. van Hooft, et al. (2004). Homeostatic scaling of neuronal excitability by synaptic modulation of somatic hyperpolarization-activated Ih channels. Proc Natl Acad Sci U S A 101(14): 5123-5128. Wahl-Schott, C. and M. Biel (2009). HCN channels: structure, cellular regulation and physiological function. Cell Mol Life Sci : CMLS 66(3): 470-494.
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Walker, M. C. and D. M. Kullmann (1999). Febrile convulsions: a 'benign' condition? Nat Med 5(8): 871-872. Wang, M., B. P. Ramos, et al. (2007). Alpha2A-adrenoceptors strengthen working memory networks by inhibiting cAMP-HCN channel signaling in prefrontal cortex. Cell 129(2): 397410. White, H. S. (2002). Animal models of epileptogenesis. Neurology 59(9 Suppl 5): S7-S14. Wierschke, S., T. N. Lehmann, et al. (2010). Hyperpolarization-activated cation currents in human epileptogenic neocortex. Epilepsia 51(3): 404-414. Wilkars, W., Z. Liu, et al. (2012). Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms. PLoS One 7(2): e32181. Williams, S. R. and G. J. Stuart (2000). Site independence of EPSP time course is mediated by dendritic Ih in neocortical pyramidal neurons. J Neurophysiol 83(5): 3177-3182. Williams, S. R. and G. J. Stuart (2003). Voltage- and site-dependent control of the somatic impact of dendritic IPSPs. J Neurosci 23(19): 7358-7367. Zolles, G., D. Wenzel, et al. (2009). Association with the auxiliary subunit PEX5R/Trip8b controls responsiveness of HCN channels to cAMP and adrenergic stimulation. Neuron 62(6): 814-825.
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Figure 1: Effects of HCN1 channels on cortical dendritic excitability. (i) Morphology of an entorhinal cortical (EC) layer III pyramidal neuron. Scale bar = 50 m (ii) Cell-attached recordings of the HCN channel current (Ih) from HCN1 null (HCN1-/-) and wildtype (wt) EC layer III dendrites. The current was generated by applying a hyperpolarizing step from -40 mV to -140 mV as shown in the schematic. (iii) Representative current-clamp recordings from wildtype and HCN1-/- EC layer III dendrites at their normal resting membrane potential (NRMP) when a series of 400 ms hyperpolarizing and depolarizing steps were applied as shown in the schematic. The scale bar shown applies to both recordings. (iv) Typical traces obtained when a 100 pA hyperpolarizing pulse was applied to EC layer III dendrites from a fixed potential of -70 mV, demonstrating that the input resistance of HCN1-/- dendrites is much greater than wildtypes. (v) Single and trains of simulated EPSPs recorded from HCN1/- and wildtype dendrites at the common potential of -70 mV in response to alpha waveform injections. Adapted from Huang et al. (2009) J. Neurosci., 29, 10979-88.
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Figure 2: Pre-synaptic HCN channels and synaptic release in the adult entorhinal cortex (EC). (i) Immunogold particles for HCN1 in assymetric synaptic terminals (presumably excitatory glutamatergic synapses) in EC layer III in wildtype (Wt). Labelling was absent in HCN1-/- tissue. Adapted from Huang et al. (2011) Nat. Neurosci., 14, 478-86. (ii) Schematic showing that pre-synaptic HCN channels and T-type Ca2+ channels are present on the same terminals. Pharmacological inhibition or genetic ablation of HCN1 channels leads to membrane hyperpolarization and enhanced Ca2+ influx through T-type Ca2+ channels,
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boosting spontaneous synaptic release.
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This is an Accepted Article that has been peer-reviewed and approved for publication in the The Journal of Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an 'Accepted Article'; doi: 10.1113/jphysiol.2013.270058. This article is protected by copyright. All rights reserved.
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Abstract The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium ion channels. They are, however, activated by hyperpolarizing potentials and are permeable to cations. Four HCN subunits have been cloned, of which HCN1 and HCN2 subunits are predominantly expressed in the cortex. These subunits are principally located in pyramidal cell dendrites, although they are also found at lower concentrations in the somata of pyramidal neurons as well as other neuron subtypes. HCN channels are actively trafficked to dendrites by binding to the chaperone protein, TRIP8b. Somato-dendritic HCN channels in pyramidal neurons modulate spike firing and synaptic potential integration by influencing the membrane resistance and resting membrane potential. Intriguingly, HCN channels are present in certain cortical axons and synaptic terminals too. Here, they regulate synaptic transmission but the underlying mechanisms appear to vary considerably amongst different synaptic terminals. In conclusion, HCN channels are expressed in multiple neuronal subcellular compartments in the cortex, where they have a diverse and complex effect on neuronal excitability.
Voltage-gated ion channels play a fundamental role in regulating neuronal activity and synaptic transmission. The abundance and biophysical properties of voltage-gated ion This article is protected by copyright. All rights reserved.
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channels varies within neuronal subcellular compartments: axons, dendrites and somata (Lai and Jan 2006; Johnston and Narayanan 2008; Nusser 2009). This variation in localization has a significant impact on neuronal and neural network excitability and thus physiological processes such as learning and memory and patho-physiological conditions such as epilepsy. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that are permeable to Na+ and K+ ions and open at potentials more negative to 50 mV (Pape 1996; Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; Shah, Hammond et al. 2010). These channels, thus, are active at the normal resting membrane potentials (RMP) of most neurons and contribute to depolarizing the RMP. In addition, HCN channels regulate the membrane resistance. By affecting both the RMP and membrane resistance, HCN channels critically influence intrinsic neuronal excitability, synaptic potential integration and neurotransmitter release (Biel, Wahl-Schott et al. 2009; Shah, Hammond et al. 2010). Four HCN subunits (HCN1-4) have so far been cloned (Ludwig, Zong et al. 1998; Santoro, Liu et al. 1998). All four are expressed in the central nervous system (CNS) but their patterns of expression vary. Of these, only HCN1 subunits are abundantly found in the cortex, hippocampus, cerebellum and brain stem (Moosmang, Biel et al. 1999; Notomi and Shigemoto 2004). In contrast, HCN2 subunits are distributed ubiquitously through the CNS, with highest expression levels in the thalamus and brain stem nuclei. HCN3 subunits are expressed at a level low in the CNS and HCN4 subunits are found in selective brain regions such as mitral cell layer of the olfactory bulb (Moosmang, Biel et al. 1999; Notomi and Shigemoto 2004). These subunits can form homomeric or heteromeric channels when expressed in heterologous systems (Chen, Wang et al. 2001; Biel, Wahl-Schott et al. 2009). The activation time constants and steady state voltagedependence of the individual HCN currents differs considerably (Biel, Wahl-Schott et al. 2009). Thus, HCN1 subunits have very fast activation kinetics whilst HCN4 subunits have the slowest kinetics. In addition, all HCN subunits are modulated by cyclic nucleotides, though the extent to which HCN1-4
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currents are modified by these varies considerably. A number of other intracellular signalling molecules such as phosphoinositides and kinases as well as auxiliary subunits such as TPR-containing Rab8b interacting protein (TRIP8b) also affect the biophysical properties and expression of HCN subunits (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; Wahl-Schott and Biel 2009; Shah, Hammond et al. 2010). This diversity in their expression, biophysical properties and modulation by intracellular molecules is, therefore, likely to differentially and dynamically regulate neuronal excitability (for in-depth reviews on HCN channel structure and biophysical properties, please see (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; Wahl-Schott and Biel 2009). In this review, I will focus on the role of HCN channels in determining cortical neuronal excitability. I will also discuss how HCN subunits are trafficked to selective subcellular compartments (axons and dendrites) and how their activity and plasticity affects pyramidal cell dendritic excitability and presynaptic function. .
Somato-dendritic HCN channel function
Electrophysiological studies have revealed that the HCN current, Ih, is greatest in the distal dendrites of cortical and hippocampal pyramidal neurons (Magee 1998; Stuart and Spruston 1998; Williams and Stuart 2000; Berger, Larkum et al. 2001; Shah, Anderson et al. 2004; Huang, Walker et al. 2009) (Fig 1). These findings have been corroborated with immunohistochemical studies showing the abundance of HCN1 and HCN2 subunits in pyramidal cell distal dendrites (Lorincz, Notomi et al. 2002; Notomi and Shigemoto 2004). HCN channels, though, are also located in the somata of some pyramidal neurons (albeit at a lower density than that in distal dendrites), interneuron subtypes and stellate cells (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009). Here, treatment with HCN channel inhibitors augments the input resistance (RN). Since the RMP is also lowered substantially, there is either a reduction or little change in the number of spikes generated in response to depolarizing stimuli (Maccaferri, Mangoni et al. 1993; Maccaferri and McBain 1996;
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Gasparini and DiFrancesco 1997; Magee 1998; Robinson and Siegelbaum 2003; Fransen, Alonso et al. 2004; Shah, Anderson et al. 2004; Aponte, Lien et al. 2006; van Welie, Remme et al. 2006; Nolan, Dudman et al. 2007; Thuault, Malleret et al. 2013). In some neurons, such as the entorhinal cortical stellate cells, HCN channels are activated during the afterhyerpolarization following spikes and thereby affect spike firing patterns (Nolan, Dudman et al. 2007). In contrast, in distal dendrites, where the expression of HCN channels is the greatest, pharmacological blockade of Ih or genetic ablation of HCN results in augmented dendritic excitability despite a hyperpolarized RMP (Magee 1998; Magee 1999; Berger, Larkum et al. 2001; Poolos, Migliore et al. 2002; Shah, Anderson et al. 2004; Huang, Walker et al. 2009)(Fig 1). This is because the increase in membrane resistance following the inhibition of HCN channels in larger in distal dendrites than at the soma such that the change in voltage induced by depolarizing stimuli in the absence of Ih results in spikes despite the RMP being hyperpolarization (Magee 1998; Poolos, Migliore et al. 2002; Shah, Anderson et al. 2004; Huang, Walker et al. 2009)(Fig 1). Changes in membrane resistance will also affect synaptic potential shapes and integration. Indeed, when HCN channel activity is reduced, somato-dendritic excitatory post-synaptic potential (EPSP) amplitudes are greater and their decay slower, leading to enhanced summation of a train of synaptic potantials (Fig 1) (Magee 1998; Magee 1999; Magee 2000; Williams and Stuart 2000; Berger, Larkum et al. 2001; Poolos, Migliore et al. 2002; Nolan, Malleret et al. 2004; Shah, Anderson et al. 2004; Huang, Walker et al. 2009; Sheets, Suter et al. 2011). Thus, trains of EPSPs are more likely to generate action potentials in axons, boosting neuronal excitability (Shah, Anderson et al. 2004). The increased EPSP summation caused by somato-dendritic HCN channel inhibition is, at least in part, due to the enhanced membrane resistance. In distal dendrites, relief of inactivation of T- and N- type Ca2+ channels by RMP hyperpolarization also contributes to enhanced EPSP summation following
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pharmacological block of HCN channels (Tsay, Dudman et al. 2007). Certainly, Ca2+ entry via these Ca2+ channels during dendritic Ca2+ spikes is greater in the presence of HCN channel inhibitors (Tsay, Dudman et al. 2007). It should be noted, though, that certain subthreshold potassium channels such as KV7/M- channels are likely to have a larger impact on EPSP summation in the absence of Ih, if expressed in the same subcellular domain within a particular neuron subtype (George, Abbott et al. 2009). Hence, the overall effect of HCN channels on EPSP summation is likely to depend to a certain extent on which other ion channels are present locally. Somato-dendritic HCN channels affect the integration of inhibitory synaptic inputs (IPSPs) too (Williams and Stuart 2003; Atherton, Kitano et al. 2010; Pavlov, Scimemi et al. 2011). HCN channels are activated by hyperpolarization and their activation during trains of IPSPs serves to limit synaptic hyperpolarization (Williams and Stuart 2003; Atherton, Kitano et al. 2010; Pavlov, Scimemi et al. 2011). In cortical pyramidal cell neurons, distal dendritic Ih enhances dendro-somatic IPSP attenuation and constrains axo-somatic depolarization (Williams and Stuart 2003). Moreover, in certain neurons, the activation of HCN channels during synaptic inhibition restricts de-inactivation of T-type Ca2+ channels and rebound action potential firing (Atherton, Kitano et al. 2010). Therefore, enhanced HCN channel activity during trains of synaptic inhibitory potentials can profoundly alter the state of neurons and their response to subsequent stimuli. Somato-dendritic HCN channels have also been implicated in regulating neuronal oscillations and subthreshold resonance properties of neurons (Fransen, Alonso et al. 2004; van Welie, Remme et al. 2006; Haas, Dorval et al. 2007; Narayanan and Johnston 2007; Nolan, Dudman et al. 2007; Narayanan and Johnston 2008; Hu, Vervaeke et al. 2009; Marcelin, Chauviere et al. 2009; Vaidya and Johnston 2013). In distal dendrites, HCN channels are thought to act as inverse leaky voltage-dependent inductors and thereby act as a
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bandpass filter to allow synaptic inputs of certain frequency to be preferentially transferred to the soma (Narayanan and Johnston 2008; Vaidya and Johnston 2013). Indeed, recent evidence suggests that HCN channels influence signal processing in distal hippocampal CA1 pyramidal dendrites such that slower, theta frequency synaptic inputs get preferentially transferred to the soma even when dendrites receive synaptic inputs at gamma frequency (Vaidya and Johnston 2013). In addition, the inductance property of HCN channels provides an efficient means of synchronization of inputs so that voltage waveforms at the soma are not very sensitive to the location of the synaptic inputs in the dendritic tree (Vaidya and Johnston 2013). Thus, HCN channels modulate somato-dendritic information processing in a variety of ways all of which impact neuronal synaptic integration and activity.
Role of pre-synaptic HCN channels in synaptic release In addition to their location in dendrites, immunohistochemical evidence has suggested that HCN1 subunits are expressed in cortical and hippocampal axons and synaptic terminals of inhibitory and excitatory neurons (Notomi and Shigemoto 2004; Lujan, Albasanz et al. 2005; Bender, Kirschstein et al. 2007; Boyes, Bolam et al. 2007; Huang, Lujan et al. 2011; Huang, Lujan et al. 2012) (Fig 2). In the rodent hippocampus, HCN channels are present in basket cell axons and terminals, where they inhibit synaptic release by as yet an undefined mechanism (Aponte, Lien et al. 2006). HCN1 subunits are also present in immature perforant path axons, which synapse onto dentate gyrus granule cells (Bender, Kirschstein et al. 2007). Here, interestingly, pharmacological inhibitors of HCN channels moderately reduced short term depression of long trains of EPSPs at 20 Hz but not at lower frequencies. These findings suggested that HCN1 subunits in immature perforant path axons form functional channels, which modulate action potential dependent release (Bender, Kirschstein et al. 2007).
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In adult neurons, the influence of HCN channels on synaptic transmission has so far only been found in the entorhinal cortex (EC). Here, immunogold labeling showed that HCN1 subunits were preferentially localized to the active zone in assymetric (presumably glutamatergic) synaptic terminals (Huang, Lujan et al. 2011; Huang, Lujan et al. 2012) (Fig 2). These terminals also contained the T-type Ca2+ channel subunits, CaV3.2, as evident from co-labelling for HCN1 and CaV3.2 subunits (Huang, Lujan et al. 2011). Pharmacological inhibition or genetic ablation of HCN1 channels resulted in enhanced frequency of miniature excitatory post-synaptic currents (mEPSCs) selectively onto EC layer III pyramidal neurons, suggesting that pre-synaptic HCN channels regulate basal glutamatergic synaptic release (Huang, Lujan et al. 2011) (Fig 2). The significantly increased mEPSC frequency was a consequence of relief of inactivation of CaV3.2 channels caused by membrane hyperpolarization when HCN channel function was reduced (Huang, Lujan et al. 2011). Interestingly, the paired pulse ratio of EPSCs evoked by stimulating distal dendrites of EC layer III pyramidal neurons was significantly lowered in the presence of HCN channel blockers, suggesting that action potential driven release onto EC layer III pyramidal neurons is also regulated by HCN channels (Huang, Lujan et al. 2011). Hence, HCN channels affect multiple modes of synaptic transmission onto EC layer III pyramidal neurons.
Dendritic and pre-synaptic HCN1 subunit trafficking. HCN channels are actively trafficked to dendrites by binding to chaperone proteins known as TPR-containing Rab8b interacting protein (TRIP8b) (Santoro, Wainger et al. 2004; Lewis, Schwartz et al. 2009; Santoro, Piskorowski et al. 2009; Zolles, Wenzel et al. 2009; Shah, Hammond et al. 2010). Moreover, TRIP8b is essential for membrane expression of HCN channels in hippocampal and cortical dendrites (Santoro, Wainger et al. 2004; Lewis, Schwartz et al. 2009; Santoro, Piskorowski et al. 2009). In addition, the presence of TRIP8b
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shifts the activation curve of Ih to the left (Lewis, Schwartz et al. 2009; Santoro, Piskorowski et al. 2009; Zolles, Wenzel et al. 2009). Moreover, the presence of TRIP8b alters sensitivity of HCN channels to cyclic nucleotides (Zolles, Wenzel et al. 2009; Han, Noam et al. 2011; Hu, Santoro et al. 2013). Thus, the presence of TRIP8b is important for the expression and voltage-dependence of dendritic HCN channels. Nine isoforms of TRIP8b have so far been identified, most of which enhance expression of dendritic HCN subunits (Lewis, Schwartz et al. 2009; Santoro, Piskorowski et al. 2009). Some TRIP8b isoforms, however, suppress HCN subunit expression (Zolles, Wenzel et al. 2009; Lewis and Chetkovich 2011; Santoro, Hu et al. 2011). Interestingly, TRIP8b isoforms that hinder HCN subunit expression have been suggested to predominantly exist in adult principal cell (including pyramidal neuron) axons (Piskorowski, Santoro et al. 2011). It has, thus, been proposed that TRIP8b prevents mislocalization of HCN subunits to adult axons (Piskorowski, Santoro et al. 2011). In agreement with this notion, HCN1 subunits are localized to immature perforant path axons when TRIP8b expression is low, suggesting that upregulation of TRIP8b expression during development leads to reduced axonal HCN subunit expression (Wilkars, Liu et al. 2012). Moreover, absence of all TRIP8b isoforms results in HCN1 expression in adult perforant path axons (Wilkars, Liu et al. 2012). HCN1 subunits, however, are expressed in certain adult axons and synaptic terminals in the entorhinal cortex (EC) (Huang, Lujan et al. 2011; Huang, Lujan et al. 2012). Are TRIP8b isoforms, therefore, not localized to these? There was no difference in HCN1 subunit expression in these axons and synaptic terminals when wildtype and TRIP8b tissue was compared (Huang, Lujan et al. 2012). Further, functional pre-synaptic HCN1 channels regulated synaptic release onto EC layer III pyramidal neurons to a similar extent in both wildtype and TRIP8b null mice, suggesting that TRIP8b is not involved in the targeting of HCN1 subunits to these axons (Huang, Lujan et al. 2012). In heterologous systems as well as
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neurons, other proteins such as filamin A have been shown to regulate HCN subunit expression (Biel, Wahl-Schott et al. 2009). It remains to be determined if these proteins might be involved in the targeting and stabilization of HCN subunits in adult axons.
Dendritic and pre-synaptic HCN channel plasticity under physiological and pathophysiological states HCN channel activity in hippocampal CA1 pyramidal neurons is modified by activitydependent mechanisms involving changes in intracellular Ca2+ concentrations (van Welie, van Hooft et al. 2004). Hebbian plasticity including NMDA-receptor dependent long-term potentiation (LTP) also alters dendritic HCN subunit expression and channel function in hippocampal CA1 pyramidal neurons (Shah, Hammond et al. 2010). In CA1 pyramidal neurons, induction of NMDA-receptor dependent LTP via a theta burst protocol enhances HCN channel expression by activating calcium/calmodulin dependent protein kinase II (CaMKII) (Fan, Fricker et al. 2005; Campanac, Daoudal et al. 2008). LTP induced by high frequency stimulation, though, reduces dendritic HCN channel function and causes greater synaptic potential summation and EPSP-spike coupling (Campanac, Daoudal et al. 2008). Further, activation of the 2 adrenoreceptors in prefrontal cortical dendritic spines led to enhanced LTP and working memory via a decrease in spine cAMP and HCN1 channel activity (Wang, Ramos et al. 2007). Alterations in modifications in Ih induced by stimulation of schaffer collateral inputs to CA1 pyramidal neurons were absent in TRIP8b null neurons, indicating changes in TRIP8b expression or function might underlie plasticity induced changes in Ih (Brager, Lewis et al. 2013). Interestingly, though, pairing of schaffer collateral and perforant path inputs produced LTP in TRIP8b null CA1 pyramidal neurons (Brager, Lewis et al. 2013). LTP induced by stimulation of the distal perforant path was also greater in HCN1 null CA1 pyramidal neurons (Nolan, Malleret et al. 2004). Consistent with this, HCN1
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null mice have increased hippocampal dependent learning and memory (Nolan, Malleret et al. 2004). In addition to LTP, induction of long-term depression (LTD) modulates Ih. Thus, metabotropic glutamate receptor-dependent LTD lowered dendritic HCN channel activity due to Ca2+ release from internal stores and activation of PKC (Brager and Johnston 2007). Therefore, whilst activity and Hebbian forms of plasticity modulate dendritic HCN channel function and expression, the consequent change in HCN channel activity (up- or downregulation) is likely to be dependent upon the locus where plasticity is induced and the intracellular signalling cascades triggered by plasticity-inducing mechanisms. Abnormal neuronal activity is a common feature of many neurological disorders too. Indeed, modifications in HCN channel expression and function have been associated with disorders such as neuropathic pain and epilepsy (Biel, Wahl-Schott et al. 2009; Noam, Bernard et al. 2011). Considerable evidence for alterations in HCN channel function has been reported in animal models of epilepsy, particularly those mimicking temporal lobe epilepsy (TLE). TLE is the most common, drug-resistant form of acquired epilepsy (Engel 1996). It can be mimicked in animals by administering chemoconvulsants such as kainic acid or pilocarpine (White 2002). Intriguingly, cortical dendritic HCN1 and HCN2 subunit expression and activity is reduced within hours of chemoconvulsant-induced status epilepticus and remains persistently downregulated for weeks (Shah, Anderson et al. 2004; Jung, Jones et al. 2007; Shin, Brager et al. 2008; Marcelin, Chauviere et al. 2009; Jung, Warner et al. 2011). There is also a long-term decrease in cortical pre-synaptic HCN channel activity following kainic acid induced status epilepticus (Huang, Lujan et al. 2012). Further, a decrease in HCN1 mRNA and current function has also been found in cortical and hippocampal tissue obtained from TLE patients (Bender, Soleymani et al. 2003; Wierschke, Lehmann et al. 2010). HCN2 mRNA levels, though, appear slightly enhanced in epileptic human hippocampus (Bender, Soleymani et al. 2003). Interestingly, though, HCN2 null mice
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have absence epilepsy (Ludwig, Budde et al. 2003) and loss of function mutations in HCN2 subunits have been associated with idiopathic generalized epilepsies in patients (DiFrancesco, Barbuti et al. 2011). HCN1 null mice, however, are not spontaneously epileptic but are more susceptible to chemoconvulsant-induced status epilepticus or kindling. (Huang, Walker et al. 2009; Santoro, Lee et al. 2010) These results suggest that the loss of HCN subunits following status epilepticus is likely to contribute to the process of epileptogenesis. The sustained reduction in HCN subunits has been attributed to multiple mechanisms including repressed transcription of HCN1 by upregulation of Neuron-Restrictive Silencer Factor (NRSF) (McClelland, Flynn et al. 2011) and altered activity of the phosphatase, calcineurin, and the kinase, p38 MAPK, resulting in a leftward shift in the HCN current activation curve and fewer HCN channels available at rest (Jung, Bullis et al. 2010). Indeed, transiently restoring HCN channel expression by disrupting the interaction between the NRSF and HCN1 delays the onset of spontaneous seizure activity following termination of SE (McClelland, Flynn et al. 2011). Intriguingly, HCN2 channel expression and function is increased in hippocampal pyramidal neurons following febrile seizures (Chen, Aradi et al. 2001; Brewster, Bender et al. 2002; Dyhrfjeld-Johnsen, Morgan et al. 2008). A heterozygous missense mutation in HCN2 (S126L) has also been correlated with increased incidence of febrile seizures (Nakamura, Shi et al. 2013). Expressed HCN2 channels containing this mutation displayed faster kinetics with higher temperatures, resulting in enhanced availability of the current under these conditions (Nakamura, Shi et al. 2013). Moreover, dendritic HCN current is enhanced in hippocampal pyramidal neurons in mice in with targeted deletions in the Fragile X FMR1 gene(Brager, Akhavan et al. 2012). FMR1 knockout mice do not exhibit spontaneous seizures but are more susceptible to audiogenic seizures (Musumeci, Bosco et al. 2000). Similarly, only about a third of the rodents subjected to febrile seizures develop chronic epilepsy
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(Walker and Kullmann 1999; Dube, Brewster et al. 2007). Hence, whether HCN channel upregulation under these conditions is a homeostatic change or cause of the epileptogenic activity remains to be further investigated.
Concluding remarks In summary, in the cortex, HCN channels are predominantly located in pyramidal neuron distal dendrites (Johnston and Narayanan 2008; Nusser 2009; Shah, Hammond et al. 2010). They are actively trafficked here by binding to TRIP8b (Shah, Hammond et al. 2010). Dendritic HCN channels lower the membrane resistance and depolarize the resting membrane potential, limiting calcium channel activation. Consequently, inhibition of these channels enhances dendritic excitability and synaptic potential summation despite the membrane potential being hyperpolarized (Robinson and Siegelbaum 2003; Shah, Hammond et al. 2010). Further, dendritic HCN channels contribute to the synchronization of synaptic potentials such that the voltage waveform at the soma is not influenced by the location of the synaptic inputs (Vaidya and Johnston 2013). In addition to TRIP8b, dendritic HCN channels are modulated by a number of different intracellular molecules including cyclic nucleotides, kinases and phosphatases (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; Shah, Hammond et al. 2010). Thus, Hebbian and homeostatic plasticity inducing mechanisms as well as pathological conditions that results in alterations of the activity of various intracellular substances modulate HCN channel function and neuronal excitability. Whilst HCN channel function in cortical pyramidal cell dendrites have been studied the most, HCN channels are also located at the somata of some pyramidal neurons, interneurons and stellate cells (Robinson and Siegelbaum 2003; Biel, Wahl-Schott et al. 2009; WahlSchott and Biel 2009) as well as in a subset of excitatory and inhibitory synaptic terminals
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(Notomi and Shigemoto 2004; Lujan, Albasanz et al. 2005; Bender, Kirschstein et al. 2007; Huang, Lujan et al. 2011). At the somata, HCN channels contribute to maintaining intrinsic excitability too. However, in contrast to their effects in dendrites, pharmacological block or genetic deletion of HCN channels at pyramidal neuron, interneuron or stellate cell somata has little effect on spike firing (Maccaferri, Mangoni et al. 1993; Maccaferri and McBain 1996; Gasparini and DiFrancesco 1997; Magee 1998; Robinson and Siegelbaum 2003; Fransen, Alonso et al. 2004; Shah, Anderson et al. 2004; Aponte, Lien et al. 2006; van Welie, Remme et al. 2006; Nolan, Dudman et al. 2007; Thuault, Malleret et al. 2013). Synaptic potential integration at the soma is affected by HCN channels as well but the overall effect on somatic excitability is likely to depend on the complement of other ion channels expressed. Similarly, HCN channels in synaptic terminals regulate synaptic release but the overall effect in the change in neurotransmission appears to be dependent on which other ion channels might also be located in particular synaptic terminals (Aponte, Lien et al. 2006; Bender, Kirschstein et al. 2007; Huang, Lujan et al. 2011; Huang, Lujan et al. 2012). In comparison to dendritic HCN channels, though, relatively little is known about the regulation, modulation and trafficking of pre-synaptic HCN channels. In summary, HCN channels are diversely expressed within the cerebral cortex, where they contribute to maintaining intrinsic neuronal activity and synaptic transmission, thereby serving to dynamically influencing cortical network activity.
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Walker, M. C. and D. M. Kullmann (1999). Febrile convulsions: a 'benign' condition? Nat Med 5(8): 871-872. Wang, M., B. P. Ramos, et al. (2007). Alpha2A-adrenoceptors strengthen working memory networks by inhibiting cAMP-HCN channel signaling in prefrontal cortex. Cell 129(2): 397410. White, H. S. (2002). Animal models of epileptogenesis. Neurology 59(9 Suppl 5): S7-S14. Wierschke, S., T. N. Lehmann, et al. (2010). Hyperpolarization-activated cation currents in human epileptogenic neocortex. Epilepsia 51(3): 404-414. Wilkars, W., Z. Liu, et al. (2012). Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms. PLoS One 7(2): e32181. Williams, S. R. and G. J. Stuart (2000). Site independence of EPSP time course is mediated by dendritic Ih in neocortical pyramidal neurons. J Neurophysiol 83(5): 3177-3182. Williams, S. R. and G. J. Stuart (2003). Voltage- and site-dependent control of the somatic impact of dendritic IPSPs. J Neurosci 23(19): 7358-7367. Zolles, G., D. Wenzel, et al. (2009). Association with the auxiliary subunit PEX5R/Trip8b controls responsiveness of HCN channels to cAMP and adrenergic stimulation. Neuron 62(6): 814-825.
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Figure 1: Effects of HCN1 channels on cortical dendritic excitability. (i) Morphology of an entorhinal cortical (EC) layer III pyramidal neuron. Scale bar = 50 m (ii) Cell-attached recordings of the HCN channel current (Ih) from HCN1 null (HCN1-/-) and wildtype (wt) EC layer III dendrites. The current was generated by applying a hyperpolarizing step from -40 mV to -140 mV as shown in the schematic. (iii) Representative current-clamp recordings from wildtype and HCN1-/- EC layer III dendrites at their normal resting membrane potential (NRMP) when a series of 400 ms hyperpolarizing and depolarizing steps were applied as shown in the schematic. The scale bar shown applies to both recordings. (iv) Typical traces obtained when a 100 pA hyperpolarizing pulse was applied to EC layer III dendrites from a fixed potential of -70 mV, demonstrating that the input resistance of HCN1-/- dendrites is much greater than wildtypes. (v) Single and trains of simulated EPSPs recorded from HCN1/- and wildtype dendrites at the common potential of -70 mV in response to alpha waveform injections. Adapted from Huang et al. (2009) J. Neurosci., 29, 10979-88.
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Figure 2: Pre-synaptic HCN channels and synaptic release in the adult entorhinal cortex (EC). (i) Immunogold particles for HCN1 in assymetric synaptic terminals (presumably excitatory glutamatergic synapses) in EC layer III in wildtype (Wt). Labelling was absent in HCN1-/- tissue. Adapted from Huang et al. (2011) Nat. Neurosci., 14, 478-86. (ii) Schematic showing that pre-synaptic HCN channels and T-type Ca2+ channels are present on the same terminals. Pharmacological inhibition or genetic ablation of HCN1 channels leads to membrane hyperpolarization and enhanced Ca2+ influx through T-type Ca2+ channels,
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boosting spontaneous synaptic release.
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